Natural Tissue Scaffolds as Tissue Fillers

ABSTRACT

Tissue fillers derived from decellularized tissues are provided. The tissue fillers can include acellular tissue matrices that have reduced inflammatory responses when implanted in a body. Also provided are methods of making and therapeutic uses for the tissue fillers.

This application claims priority under 35 U.S.C. §§119-120 to U.S.patent application Ser. No. 13/560,362, which was filed on Jul. 27,2012, and to U.S. Provisional Application No. 61/512,610, filed on Jul.28, 2011, which are incorporated herein by reference in its entirety.

The present disclosure relates generally to tissue fillers and their useas implants and scaffolds for natural tissue regrowth after removal of aportion of native tissue.

Currently, tissue fillers are often derived from temporary hyaluronicacid or collagen-based materials. These materials lack stability andbiocompatibility, and may require complex harvesting procedures. Theirmedical use is generally limited to temporarily filling small sites oftissue removal. Thus, existing tissue fillers are not suitable for longterm removal of large volumes of tissue, such as breast lumpectomies. Inaddition, existing tissue fillers may not promote sufficient nativetissue regrowth or limit inflammation and the formation of scar tissue.Producing a tissue filler having the texture and structural integrity ofnative tissue that is also capable of promoting the regrowth of nativetissue while reducing inflammation and the formation of scar tissuewould therefore be desirable.

Accordingly, improved tissue fillers are provided herein. In variousembodiments, a tissue filler is provided, comprising an acellular tissuematrix and at least one of exogenous hyaluronic acid (HA) and exogenousdecorin at a concentration sufficient to reduce an inflammatory responseor fibrosis, when the tissue filler is implanted in a body. Theacellular tissue matrix can be selected from an acellular lung, liver,bladder, muscle, and fat matrix. In further embodiments, theconcentration of HA on the acellular tissue matrix is betweenapproximately 0.5 mg and approximately 5.0 mg per gram of tissue filler.In further embodiments, the concentration of decorin on the acellulartissue matrix is between approximately 0.3 mg and approximately 1.0 mgper gram of tissue filler. In still further embodiments, the tissuefiller elicits a reduced inflammatory response, as compared to a tissuefiller lacking HA and/or decorin, when implanted in the body. In furtherembodiments, the tissue filler reduces fibrosis and scar tissueformation after removal of a native tissue, as compared to a tissuefiller lacking HA and/or decorin, when implanted in the body.

In various embodiments, the tissue filler further comprises at least onegrowth factor. In further embodiments, the at least one growth factor isFGF, VEGF, PDGF, angiopoitin-2, or follistatin. In some embodiments, thetissue filler lacks substantially all alpha-galactose moieties. Incertain embodiments, the tissue filler has been treated to reduce abioburden. In further embodiments, the tissue filler is sterile.

In some embodiments, the tissue filler further comprises anantimicrobial agent. The antimicrobial agent can include at least one ofCHX and silver. The CHX can be at a concentration of betweenapproximately 0.1 mg and approximately 3.0 mg per gram of tissue filler.The silver can be at a concentration of between approximately 0.1 mg andapproximately 1.0 mg per gram of tissue filler.

In various embodiments, the tissue filler is compressible. In furtherembodiments, the tissue filler is capable of being compressed up toapproximately ⅔ of its length or width. In still further embodiments,the tissue filler is capable of returning to its original dimensionsafter release of compression.

In various embodiments, a method of treating a tissue after removal ofnative tissue is provided, comprising implanting the tissue fillerdescribed above into the tissue. In further embodiments, the implantedtissue filler can swell to fill a region of native tissue that has beenremoved. In still further embodiments, the implanted tissue filler isselected to have the same structural strength, texture and feel as thenative tissue it replaces. In even further embodiments, implanting thetissue filler promotes the infiltration, migration, growth, and/orproliferation of surrounding native tissue cells in the tissue filler,as well as the revascularization of the tissue being treated.

In certain embodiments, the HA and/or decorin on the implanted tissuefiller elicits a reduced inflammatory response, as compared to animplanted tissue filler lacking HA and/or decorin. In furtherembodiments, the inflammatory response is reduced by at least 10%. Instill further embodiments, the HA and/or decorin on the implanted tissuefiller reduces scar tissue formation after removal of a native tissue,as compared to an implanted tissue filler lacking HA and/or decorin. Ineven further embodiments, the decorin and/or HA remains on the tissuefiller for the duration of the implant.

In some embodiments, the method of treating a tissue further comprisingremoving at least 20% by mass of a native tissue prior to implanting atissue filler. In certain embodiments, the tissue being removedcomprises a tumor. In some embodiments, the tissue being removedcomprises breast tissue.

In various embodiments, a method of preparing a tissue filler isprovided, comprising selecting a tissue, decellularizing the tissue, andcontacting the tissue with least one substance that reduces inflammationand/or fibrosis when the tissue is implanted in a body. The tissue canbe selected from lung, liver, bladder, muscle, and fat. In someembodiments, the at least one substance that can reduce inflammationand/or fibrosis includes at least one of hyaluronic acid (HA) anddecorin. In some embodiments, when HA is used, the method includescontacting the tissue filler with a solution containing HA at aconcentration of between approximately 1.0 mg/ml and approximately 10.0mg/ml. In some embodiments, when decorin is used, the method includescontacting the tissue filler with a solution containing decorin at aconcentration of between approximately 0.1 mg/ml and approximately 3.0mg/ml.

In various embodiments, the method of preparing a tissue fillercomprises contacting the tissue filler with at least one detergent. Theat least one detergent can include at least one of sodium dodecylsulfate, sodium deoxycholate, and Triton X-100. The detergentconcentration and detergent exposure time can be selected to preventremoval of growth factors from the tissue filler. In some embodiments,the method of preparing a tissue filler further comprises removingalpha-galactose moieties from the tissue filler.

In some embodiments, the method of preparing a tissue filler comprisesirradiating the tissue filler to reduce the bioburden of the tissuefiller. Irradiation can include exposing the tissue filler to 15-17 kGyE-beam irradiation. In certain embodiments, the method further comprisessterilizing the tissue filler. In some embodiments, the method comprisescontacting the tissue filler with an antimicrobial agent. Theantimicrobial agent can include at least one of CHX and silver. In someembodiments, CHX is present at a concentration of between approximately0.1 mg and approximately 3.0 mg of CHX per gram of tissue filler. Inother embodiments, silver is present at a concentration of betweenapproximately 0.1 mg and approximately 1.0 mg of silver per gram oftissue filler.

In some embodiments, the method of preparing a tissue filler comprisesfreeze-drying the tissue filler. In further embodiments, the methodcomprises rehydrating the freeze-dried tissue filler prior toimplantation in a tissue.

In various embodiments, a tissue filler is provided, prepared by themethods described above.

In various embodiments, a method of treatment is provided, comprisingremoving a native tissue and implanting a tissue filler, wherein thetissue filler comprises an acellular tissue matrix and at least one ofexogenous hyaluronic acid (HA) and exogenous decorin at a concentrationsufficient to reduce an inflammatory response or fibrosis when thetissue filler is implanted in a body. In some embodiments, the nativetissue being removed comprises a tumor. In certain embodiments, thenative tissue is breast tissue. In some embodiments, the tissue fillerused in the method of treatment is prepared according to any one of themethods described above. In some embodiments, the tissue filler used inthe method of treatment comprises any one of the tissue fillersdescribed above.

In various embodiments, a method of enhancing a native tissue isprovided, comprising implanting a tissue filler into a native tissue,wherein the tissue filler comprises an acellular tissue matrix and atleast one of exogenous hyaluronic acid (HA) and exogenous decorin at aconcentration sufficient to reduce an inflammatory response or fibrosiswhen the tissue filler is implanted in a body. In further embodiments,the native tissue is breast tissue.

DESCRIPTION OF THE DRAWINGS

FIG. 1A shows fresh porcine lung stained with hematoxylin and eosin(H&E). FIG. 1B shows processed porcine lung stained with H&E. FIG. 1Cshows processed porcine lung stained with Verhoeff's stain. FIG. 1D is ascanning electron micrograph of processed porcine lung, as producedaccording to certain embodiments.

FIG. 2A shows collagen type-I immunostaining of acellular porcine lung.FIG. 2B shows collagen type-IV immunostaining of acellular porcine lung.FIG. 2C shows fibronectin immunostaining of acellular porcine lung, andFIG. 2D shows collagen type-III immunostaining of acellular porcinelung.

FIG. 3A is an illustration of the compressibility of acellular porcinelung. FIG. 3B is an illustration of the elasticity of acellular porcinelung after the release of compression.

FIG. 4A illustrates the shape of a porcine lung tissue filler beforefreeze drying. FIG. 4B illustrates the shape of a porcine lung tissuefiller after freeze drying. FIG. 4C illustrates the shape of a porcinelung tissue filler after rehydration.

FIG. 5 is a plot of the glycosaminoglycan (GAG) concentration inextracellular matrices derived from porcine vessel, dermal, liver, andlung tissue.

FIG. 6 shows thermogram plots for tissue fillers derived from porcinelung and liver.

FIG. 7 is a plot showing the effect of collagenase digestion on tissuefillers derived from porcine liver, lung, and dermal tissue.

FIG. 8A shows alcian blue staining of tissue filler derived from porcinelung. FIG. 8B shows alcian blue staining of tissue filler derived fromporcine lung that is coated in hyaluronic acid.

FIG. 9 is a plot showing the concentration of HA in uncoated porcineliver tissue filler (left), in porcine liver tissue filler incubatedwith 5 mg/ml of hyaluronic acid sodium salt (right), and in porcineliver tissue filler incubated with 5 mg/ml of hyaluronic acid sodiumsalt and washed overnight (center).

FIG. 10A shows anti-human decorin staining of tissue filler derived fromporcine liver. FIG. 10B shows control serum staining of tissue fillerderived from porcine liver that is coated in human decorin. FIG. 10Cshows anti-human decorin staining of tissue filler derived from porcineliver that is coated in human decorin.

FIG. 11 is a plot showing the concentration of decorin in uncoatedporcine liver tissue filler (left) and in porcine liver tissue fillerincubated with 1 mg/ml of decorin and washed overnight (right).

FIG. 12A is an H&E stain showing in vitro growth of rat fibroblast intissue fillers derived from porcine lung. FIG. 12B is an H&E stainshowing in vitro growth of rat fibroblast in tissue fillers derived fromporcine liver. FIG. 12C is an H&E stain showing in vitro growth of ratstem cells in tissue fillers derived from porcine lung. FIG. 12D is anH&E stain showing in vitro growth of rat stem cells in tissue fillersderived from porcine liver.

FIG. 13 illustrates inflammatory cytokine levels induced by in vitroculturing of human blood mononuclear cells with tissue fillers derivedfrom porcine lung, liver and dermal tissue.

FIG. 14A illustrates the gross morphology of tissue fillers derived fromporcine lung two weeks after implantation in rat. FIG. 14B illustratesthe gross morphology of tissue fillers derived from porcine liver twoweeks after implantation in rat. FIG. 14C illustrates the grossmorphology of tissue fillers derived from porcine liver and coated inCHX, two weeks after implantation in rat. FIG. 14D illustrates the grossmorphology of tissue fillers derived from porcine liver and coated inhyaluronic acid, two weeks after implantation in rat. FIG. 14Eillustrates the gross morphology of tissue fillers derived from porcineliver and coated in decorin, two weeks after implantation in rat.

FIG. 15A shows H&E staining of tissue fillers derived from porcineliver. FIG. 15B shows H&E staining of tissue fillers derived fromporcine liver coated in chlorhexidine (CHX). FIG. 15C shows H&E stainingof tissue fillers derived from porcine liver coated in hyaluronic acid.FIG. 15D shows H&E staining of tissue fillers derived from porcine livercoated in decorin.

FIG. 16A shows H&E staining of tissue fillers derived from porcineliver. FIG. 16B shows H&E staining of tissue fillers derived fromporcine liver coated in chlorhexidine (CHX). FIG. 16C shows H&E stainingof tissue fillers derived from porcine liver coated in hyaluronic acid.FIG. 16D shows H&E staining of tissue fillers derived from porcine livercoated in decorin.

FIG. 17A shows H&E staining of tissue fillers derived from porcine livercoated in HA. FIG. 17B shows H&E staining of tissue fillers derived fromporcine liver coated in decorin.

FIG. 18A shows immunostaining of fibroblast cells in a porcine livertissue filler two weeks after implantation in rat. FIG. 18B showsanti-vWF immunostaining of neo-vessel formation in a porcine livertissue filler two weeks after implantation in rat. FIG. 18C showsanti-SMC-α-actin immunostaining of myofibroblast cells in a porcineliver tissue filler two weeks after implantation in rat.

FIG. 19A shows anti-vimentin immunostaining of fibroblast cells in aporcine liver tissue filler two weeks after implantation in rat. FIG.19B shows anti-vimentin immunostaining of fibroblast cells in a porcineliver tissue filler coated in hyaluronic acid, two weeks afterimplantation in rat. FIG. 19C shows anti-vimentin immunostaining offibroblast cells in a porcine liver tissue filler coated in decorin, twoweeks after implantation in rat.

FIG. 20A shows anti-human decorin immunostaining of tissue fillersderived from porcine liver two weeks after implantation in rat. FIG. 20Bshows control serum staining of tissue fillers derived from porcineliver coated in human decorin, two weeks after implantation in rat. FIG.20C shows anti-human decorin immunostaining of a tissue filler derivedfrom porcine liver and coated in human decorin, two weeks afterimplantation in rat.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Reference will now be made in detail to certain exemplary embodimentsaccording to the present disclosure, certain examples of which areillustrated in the accompanying drawings.

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. Also in this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including,” as well as other forms, such as “includes” and “included,”are not limiting. Any range described herein will be understood toinclude the endpoints and all values between the endpoints.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose. To the extent publications and patentsor patent applications incorporated by reference contradict theinvention contained in the specification, the specification willsupersede any contradictory material.

In various embodiments, tissue fillers are provided. The tissue fillercan comprise an acellular tissue matrix and at least one substance thatcan reduce inflammation and/or fibrosis when the filler is implanted inthe body. The substance can be hyaluronic acid (HA) and/or decorin, at aconcentration sufficient to reduce inflammation or fibrosis afterimplantation in the body.

The acellular tissue matrix in a tissue filler can be derived fromvarious organ sources. Organs having a compact three dimensional shapeare preferable, such as lung, liver, bladder, muscle, or fat, as theyprovide a spongy acellular matrix after decellularization. This spongytissue filler can be molded and used as an implant to fill the void leftby the removal of native tissue. In certain embodiments, the acellulartissue matrix is an acellular lung or liver tissue matrix. In someembodiments, the acellular tissue matrix is an acellular porcine lung orliver tissue matrix.

In various embodiments, the tissue fillers are useful as implantsfollowing removal of native tissue from a recipient organ or tissue, oras implants for cosmetic enhancement purposes. Tissue fillers derivedfrom organs such as lung, liver, bladder, muscle, or fat provide thetexture and structural strength of native tissue, while also providing abiological scaffold in which native cells and vasculature can migrateand proliferate. Furthermore, adding at least one substance such as HAand/or decorin to the acellular tissue can help reduce undesirableinflammation and/or fibrosis following implantation of a tissue fillerinto a recipient organ or tissue.

In certain embodiments, tissue fillers can be produced bydecellularizing an organ tissue and coating the tissue in a solutioncontaining an anti-inflammatory and/or an anti-fibrotic substance suchas HA and/or decorin. In other embodiments, the presently describedtissue fillers can be further processed into desired shapes and storedeither fresh or freeze-dried prior to implantation in a recipient organ.The tissue fillers can be produced as aseptic or sterile materials. Insome embodiments, the tissue filler is in strips, balls, or molded intoother shapes that provide the desired size, shape, or structuralfeatures necessary for a given tissue filler.

As noted, the tissue fillers can comprise acellular tissue matrices,providing natural tissue scaffolds on which native tissue can grow andregenerate. As used herein, “native” cells or tissue means the cells ortissue present in the recipient organ or tissue prior to implantation ofa tissue filler. Tissue fillers derived from organs having a compactthree dimensional shape, such as lung, liver, bladder, muscle, or fat,are preferable as they provide a spongy acellular tissue matrix afterdecellularization. These spongy acellular matrices can be molded to fillthe void left by removal of a native tissue while providing the textureand durability of native tissue. Furthermore, the acellular tissuematrix provides a natural tissue scaffold for native cell growth. Thescaffold may consist of collagen, elastin, or other fibers, as well asproteoglycans, polysaccharides and growth factors. Tissue fillers mayretain all components of the extracellular matrix, or variousundesirable components may be removed by enzymatic or genetic meansprior to implantation. The exact structural components of theextracellular matrix will depend on the tissue selected and theprocesses used to prepare the tissue scaffold. The natural tissuescaffold in a tissue filler provides a structural network of fibers,proteoglycans, polysaccharides, and growth factors on which nativetissue and vasculature can migrate, grow, and proliferate.

Tissue fillers can contain acellular tissue matrices derived fromvarious tissues and animal sources. In certain embodiments, theacellular tissue is taken from human cadaver, cow, horse, or pig. Insome embodiments, tissue fillers contain acellular tissue matricesderived from lung, liver, bladder, muscle, or fat tissue in order toapproximate the soft and spongy property of a native soft tissue. Insome embodiments, the tissue is acellular porcine liver or lung tissue.

In various embodiments, tissue fillers comprise at least one substancethat can reduce inflammation and/or fibrosis after implantation into thebody (i.e., into a recipient tissue or organ), as compared to tissuematrices lacking such substances. In certain embodiments, the substanceis exogenous HA and/or exogenous decorin. Decorin is a proteoglycancommonly found in connective tissue and implicated in fibrilogenesis. HAis a glycosaminoglycan commonly found in epithelial tissue. In furtherembodiments, the hyaluronic acid is at a concentration of, e.g., 0.5,1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg of hyaluronic acid pergram of tissue filler (or any value in between). In other embodiments,the decorin is at a concentration of, e.g., 0.3, 0.35, 0.4, 0.45, 0.5,0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0 mg of decorinper gram of tissue filler (or any value in between). In someembodiments, tissue fillers containing decorin and/or HA can reduceinflammation and/or fibrosis after implantation in a recipient tissue,as compared to an implanted tissue filler lacking HA and/or decorin.

In some embodiments, the tissue fillers contain natural tissuescaffolds, and these can be used to replace the tissue scaffold lostafter removal of native tissue. Any natural tissue scaffold can be usedthat approximates the consistency, texture, or structural integrity ofthe native tissue that it is replacing. The texture and structuralproperties of a given tissue filler will depend on the tissue sourceselected, as well as on the method chosen to process the harvestedtissue. In various embodiments, lung, liver, bladder, muscle, or fattissue is used to provide effective natural tissue scaffolds becausethey provide the consistency, biocompatibility, and structural integrityof native tissue. In some embodiments, lung or liver tissue is used.

In various embodiments, the tissue fillers are compressible. Forexample, the tissue fillers may be compressed up to approximately ⅔ oftheir initial length or width. In still further embodiments, the tissuefiller is capable of returning to its original dimensions after therelease of compression. In even further embodiments, a tissue fillerderived from decellularized porcine lung is capable of being compressedup to approximately ⅔ of its length or width and then returning tosubstantially the same original dimensions after the release ofcompression (see FIG. 3). In various embodiments, lung or liver tissueis used as a tissue filler because, after decellularization andsubsequent processing (as described below), the filler exhibitssubstantial ability to stretch or compress.

Tissue fillers derived from lung and liver contain abundantglycosaminoglycans when compared to other porcine tissues. In addition,in certain embodiments, tissue fillers derived from lung and livertissue retain the major growth factors present in unprocessed lung orliver tissue. For example, lung and liver tissue fillers can retain FGF,VEGF, PDGF, angiopoitin-2 and/or follistatin (among other growthfactors). In certain embodiments, 10, 20, 30, 40, 50, or 60 ng of FGFare present per gram of dried tissue filler (or any value in between).In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng of VEGF arepresent per gram of dried tissue filler (or any value in between). Insome embodiments, 0.5, 1.0, 1.5, or 2.0 ng of PDGF are present per gramof dried tissue filler (or any value in between). In some embodiments,0.05, 0.1, or 0.2 ng of angiopoitin-2 are present per gram of driedtissue filler (or any value in between) where the tissue filler is madefrom processed lung tissue. In some embodiments, 0.5, 1.0, or 1.5 ng offollistatin are present per gram of dried tissue filler (or any value inbetween).

In certain embodiments, the tissue fillers lack certain antigens. Forexample, certain animal tissues contain alpha-galactose (α-gal) epitopesthat are known to elicit reactions in humans. Therefore, tissue fillersproduced from animal tissues can be produced or processed to lackcertain antigens, such as α-gal. In some embodiments, tissue fillerslack substantially all α-gal moieties. Elimination of the α-gal epitopesfrom the natural tissue scaffold may diminish the immune responseagainst the tissue filler, as the α-gal epitope is absent in humans. U.Galili et al., J. Biol. Chem. 263: 17755 (1988). Since non-primatemammals (e.g., pigs) produce α-gal epitopes, xenotransplantation oftissue filler material from these mammals into primates may result inrejection because of primate anti-Gal binding to the α-gal epitopes onthe tissue filler material. The binding results in the destruction ofthe tissue filler material by complement fixation and byantibody-dependent cell cytotoxicity. U. Galili et al., Immunology Today14: 480 (1993); M. Sandrin et al., Proc. Natl. Acad. Sci. USA 90: 11391(1993); H. Good et al., Transplant. Proc. 24: 559 (1992); B. H. Collinset al., J. Immunol. 154: 5500 (1995).

As described in detail below, in various embodiments, the tissuesfillers can be processed to remove antigens such as α-gal, e.g., byenzymatic treatment. Alternatively, the tissue fillers can be producedfrom animals that have been genetically modified to lack those epitopes.

In various embodiments, tissue fillers have reduced bioburden (i.e., areduced number of microorganisms growing on the tissue filler). In someembodiments, tissue fillers lack substantially all bioburden (i.e., thetissue fillers are aseptic or sterile). In certain embodiments, thetissue fillers further comprise an antimicrobial agent to eliminatemicrobial growth and/or prevent microbial growth when implanted. Theantimicrobial agent can include, for example, chlorhexidine (CHX) orsilver. In certain embodiments, the concentration of CHX or silver isadjusted to remove substantially all bioburden and/or to preventmicrobial growth. Effective concentrations of CHX capable ofsubstantially reducing bioburden on tissue fillers may include 0.1 mg,0.5 mg, 0.7 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, or 3.0 mg pergram of tissue filler (or any value in between). Effectiveconcentrations of silver capable of substantially reducing bioburden ontissue fillers may include 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6mg, 0.7 mg, 0.8 mg, 0.9 mg, or 1.0 mg per gram of tissue filler (or anyvalue in between). As used herein, “substantially all bioburden” meanstissue fillers in which the concentration of microorganisms growing onthe filler is less than 1%, 0.1%, 0.01%, 0.001%, or 0.0001% of thatgrowing on untreated fillers.

The tissue fillers, as described above, can be used after surgicalremoval of native tissue and/or to augment existing native tissues. Incertain embodiments, the method of use comprises removing a nativetissue and implanting a tissue filler. As used herein, the “nativetissue” being removed can be a portion, fragment, or entirety of atissue found in a body. The tissue filler can comprise an acellulartissue matrix and at least one substance capable of reducinginflammation and/or fibrosis. In certain embodiments, the substance isexogenous HA and/or exogenous decorin at a concentration sufficient toreduce an inflammatory response when the tissue filler is implanted inthe body. In various embodiments, the tissue filler used after removalof a native tissue comprises the tissue filler described above, or isprepared as described below. In some embodiments, the tissue beingremoved comprises a tumor. In certain embodiments, the tissue is breasttissue.

In various embodiments, the tissue fillers described above are usedafter removal of a native soft tissue because they have a sponge-likeconsistency and can swell to fill the region of tissue that has beenremoved. In addition, the tissue fillers can retain the structuralstrength, texture, and/or feel of native soft tissue. Thus, the tissuefillers can preserve the shape of the excised natural tissue. Forinstance, tissue fillers derived from lung or liver can be compressed byup to approximately ⅔ of their length or width before returning to theiroriginal shape after release of the compressing force, therebysimulating the texture and elasticity of the native soft tissue that hasbeen removed. Furthermore, in certain embodiments, tissue fillerscontaining anti-inflammatory and/or anti-fibrotic substances can reduceinflammation and/or scar tissue formation following implantation into arecipient organ. In various embodiments, the anti-inflammatory and/oranti-fibrotic substances are HA and/or decorin.

In certain embodiments, tissue fillers produced from lung, liver,bladder, muscle, or fat tissue are implanted after removal of nativetissue. In some embodiments, these tissue fillers are rich in elastinand collagen, as well as glycosaminoglycans and growth factors. Thetissue fillers thus provide natural tissue scaffolds that approximatethe structure, texture, and cellular growth conditions of the tissuethat has been removed. In further embodiments, porcine lung or livertissue is used because its extracellular matrix has a well-organizedsponge structure that approximates the texture of a native soft tissue.

In various embodiments, tissue fillers produced from lung, liver,bladder, muscle, or fat tissue do not elicit a significant inflammatoryresponse when implanted in a tissue, as compared to implants derivedfrom other tissues or from non-biologic sources. For example, tissuefillers produced from lung or liver tissue do not induce significantincreases in cytokine secretion after implantation when compared tissuefillers produced from other tissue sources, such as dermis. Suitablecytokines to measure in evaluating the inflammatory response includeIL-1, IL-6, IL-8, or IL-10. Similarly, any other indicator of theinflammatory response known to one of skill can be used to measure theinflammatory response. The inflammatory response can be assayed byvarious techniques, including in vitro incubation of tissue fillers withmononuclear blood cells or in vivo implantation of tissue fillers into ahost tissue. Either method may involve cytokine immune staining ordirect cytokine quantification. Other suitable techniques for assayingthe inflammatory response are known in the art and may be used.

To further reduce inflammation after implantation into a recipienttissue, tissue fillers can be coated with or otherwise containsubstances that reduce inflammation. As used herein, a “coated” tissuefiller is one that has been contacted with an anti-inflammatory reagentor a solution containing the anti-inflammatory reagent on one or moresurfaces of the tissue filler or within the tissue filler. For example,tissue fillers can be coated in decorin and/or hyaluronic acid (HA). Insome embodiments, tissue fillers containing decorin and/or HA reduceinflammation after implantation in a tissue, as compared to an implantedtissue filler lacking HA and/or decorin. In further embodiments, coatinga tissue filler in decorin and/or HA reduces inflammatory T cell, Bcell, and/or macrophage infiltration into the tissue by 10%, 20%, 30%,40%, 50%, 60%, 70%, or 80% (or any value in between) after implantationinto a recipient organ, as compared to infiltration after an uncoatedtissue filler is implanted. In further embodiments, the decorin and/orHA coating remains on the tissue filler for the duration of the implant.In still further embodiments, the coating of decorin and/or HA remainson the tissue filler 5 days, 10 days, 15 days, 20 days, or 25 days, 1month, or 2 months (or any value in between) after implantation in atissue.

A major challenge after removal of native tissue is the formation ofundesirable scar tissue. After excision of a large volume of tissue,dense fibrosis will form due to the cross-linking of collagen duringwound healing. Preventing or reducing the amount of scar tissue thatforms after bulk tissue removal is therefore desirable in order topreserve the appearance and texture of the tissue. Thus, in variousembodiments, tissue fillers are first coated in anti-fibrosis reagentsbefore being implanted in a tissue, thereby reducing the amount of scartissue formed after implantation. As used herein, a “coated” tissuefiller is one that has been contacted with an anti-fibrosis reagent or asolution containing the anti-fibrosis reagent on one or more surfaces ofthe tissue filler or within the tissue filler.

In certain embodiments, the anti-fibrosis reagent is HA or decorin or acombination of the two. For example, decorin can stabilize the structureof native cell colonies growing in the tissue filler while preventingthe infiltration of excessive fibrous connective tissue. HA and/ordecorin can also reduce the inflammation caused by implantation of atissue filler, thereby further reducing the amount of scar tissueformation. In further embodiments, coating a tissue filler in decorinand/or HA reduces fibrosis by 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%(or any value in between) after implantation into a recipient organ, ascompared to fibrosis after an uncoated tissue filler is implanted. Infurther embodiments, the decorin and/or HA coating remains on the tissuefiller for the duration of the implant. In still further embodiments,the coating of decorin and/or HA remains on the tissue filler 5 days, 10days, 15 days, 20 days, or 25 days, 1 month, or 2 months (or any valuein between) after implantation in a tissue.

In certain embodiments, tissue fillers produced from lung or livertissue are more resistant to collagenase digestion after implantation ina tissue when compared to tissue fillers produced from dermal tissue.Collagenases are enzymes which break the peptide bonds in collagen andcan therefore degrade the extracellular matrix structures of implantedtissue fillers. Thus in some embodiments, tissue fillers produced fromlung or liver tissue provide natural tissue scaffolds that retain theirstructural integrity and ability to promote native cell repopulation forlonger periods of time after implantation when compared to tissuefillers derived from other tissue sources, such as dermal tissue.

In certain embodiments, tissue fillers retain their shape and structuralintegrity after implantation for one week, two weeks, three weeks, 1month or 2 months (or any time period in between). In other embodiments,the tissue fillers retain their structural integrity for the duration oftherapeutic use.

The ability of tissue fillers derived from tissue sources such as lung,liver, bladder, muscle, or fat to hold and retain desired shapes (due totheir spongy extracellular matrix structure) allows for the use oftissue fillers after removal of large tissue defects (e.g., large tumorremovals). For example, the removal of more than 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, or 90% (or any percentage in-between) of a tissuemass creates a large void that requires the use of a filler to replacethe lost tissue and to provide structural integrity for the remainingtissue or organ. Existing filler substances are generally used only tofill in small areas (e.g., as cosmetic fillers to remove skin wrinkles)and also lack the durability and biocompatibility needed after removalof a large volume of native tissue. In certain embodiments, a tissuefiller as described above is implanted after removal of more than 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (or any percentage in-between)of native tissue. The spongy extracellular matrix of the tissue fillersallows them to fill the void left by the removed tissue, providing abiocompatible and durable scaffold that persists for a sufficient lengthof time to enable the migration and proliferation of native tissue andvasculature into the scaffold. In some embodiments, the tissue beingremoved and replaced with tissue filler is breast tissue. In otherembodiments, the tissue being removed is muscle. In still furtherembodiments, the tissue being removed is liver tissue.

Tissue fillers that can provide structural support are useful to preventvisible detection of a tissue removal, i.e. for aesthetic purposes.Similarly, a tissue filler having a consistency similar to that of aremoved soft tissue allows the tissue filler to provide the texture andfeel the tissue had before surgical removal. For example, bulk tissueremoval in breast cancer results in a disfigured breast. Implanting atissue filler having the structural strength and elasticity of naturalbreast tissue can assist in breast reconstruction after surgery. Thus,in some embodiments, tissue fillers are used as implants after removalof a native soft tissue in order to retain the appearance and feel thetissue had prior to soft tissue removal. In further embodiments, thetissue removed is a tumor. In further embodiments, the tumor is a breasttumor.

In other embodiments, tissue fillers are implanted after loss or removalof large amounts of muscle tissue, for example due to muscle wasting ortumor growth. In other embodiments, tissue fillers are used as naturaltissue scaffolds for liver repair or regeneration after liver resection.

In various embodiments, implanting a tissue filler can promote therepair or regeneration of a tissue after bulk tissue removal. Tissuerepair or regeneration can include, for example, the infiltration ofnative cells from the surrounding tissue into the natural tissuescaffold of the tissue filler. Other examples of tissue repair andregeneration include the growth and proliferation of native cells in thenatural tissue scaffold. In some embodiments, the tissue filler is ableto support at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of the cellproliferation supported by the native tissue. Still further examples oftissue repair and regeneration include revascularization of the tissuein the region where native tissue has been removed. In furtherembodiments, the preservation of growth factors, including FGF, VEGF,PDGF, angiopoitin-2 or follistatin (among others), in tissue fillers canenhance angiogenesis or revascularization at the implant site. Incertain embodiments, implanting a tissue filler derived from lung,liver, bladder, muscle, or fat leads to fibroblast cell infiltration,growth, and/or proliferation. In further embodiments, implanting atissue filler derived from lung, liver, bladder, muscle, or fat leads toneo-vessel formation.

In certain embodiments, tissue fillers as disclosed herein can also beused outside the reconstruction context. For example, tissue fillers canbe used as implants for aesthetic enhancement purposes. Tissue fillerscan be implanted into a native tissue to enhance the shape, look, orfeel of a native tissue. The aesthetic tissue targets can includebreast, lip, cheek, and buttocks implants, among others. In certainembodiments, implanting tissue fillers for aesthetic purposes can alsolead to native tissue cell infiltration, growth, proliferation, orvascularization of the implanted tissue filler.

Production of Tissue Fillers

Tissue fillers can be produced by processing tissue from various animalsources. In certain embodiments, the tissue is taken from human cadaver,cow, horse, or pig. In some embodiments, the tissue is lung, liver,bladder, muscle, or fat tissue. In further embodiments, the tissue isporcine lung or liver tissue. In some embodiments, an entire organ isused to prepare a tissue filler. In other embodiments, portions of theorgan are processed into tissue fillers. In further embodiments, theorgan portions may include strips, balls, or other tissue fragments thatprovide the desired size, shape, or structural features necessary for agiven tissue filler.

Tissue can be subjected to multiple rounds of freeze/thaw to disrupt thetissue and improve the decellularization process. In addition, bronchior large blood vessels can be removed from tissue by manual dissection,and the tissue can be washed to remove blood cells. Any suitable washingsolution can be used, including distilled water, HEPES buffer, orphosphate buffered saline, among others.

Next, the tissue is decellularized in order to remove cells from theremaining natural tissue scaffold. Various detergents can be used todecellularize, including sodium dodecyl sulfate, sodium deoxycholate,and Triton X-100. Other examples of suitable decellularizationdetergents and decellularization procedures are described in Cortiellaet al, Tissue Engineering 16: 2565-2580 (2010), hereby incorporated inits entirety.

The concentration of detergent used to decellularize can be adjusted inorder to preserve desirable matrix proteins and prevent protein damageduring the decellularization process. The detergent may be used, forexample, at a concentration of approximately 0.1%, 0.2%, 0.5%, 1%, 2%,3%, 4%, or 5% and tissue can be incubated with detergent for 2 hours, 3hours, 5 hours, 10 hours, 12 hours, 24 hours, 2 days, 3 days, 5 days, 7days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks (or anyconcentration or time period in between). Appropriate concentrations andduration of detergent incubation can be adjusted depending on thedetergents used and the desired harshness of decellularization.

In certain embodiments, the concentration of detergents and/or detergentincubation times are reduced in order to retain a higher level of growthfactors in the extracellular matrix after decellularization. Forexample, a less harsh decellularization process may lead to theretention of increased levels of FGF, VEGF, PDGF, angiopoitin-2, andfollistatin. In another example, the concentration of detergents and/orincubation times are increased in order to more completely remove cellsfrom the extracellular matrix. Further, decellularization can beperformed so as to remove substantially all viable cells from theextracellular matrix. As used herein, “substantially all viable cells”means tissue fillers in which the concentration of viable cells is lessthan 1%, 0.1%, 0.01%, 0.001%, or 0.0001% of the cells found in thetissue or organ from which the tissue filler is made.

In certain embodiments, the major growth factors present in unprocessedtissue are preserved by the decellularization process. For example, FGF,VEGF, PDGF, angiopoitin-2 and/or follistatin can be preserved in thedecellularized tissue. In some embodiments, 10, 20, 30, 40, 50, or 60 ngof FGF are present after decellularization per gram of dried tissuefiller (or any value in between). In some embodiments, 1.0, 2.0, 3.0,4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 ng of VEGF are present afterdecellularization per gram of dried tissue filler (or any value inbetween). In some embodiments, 0.5, 1.0, 1.5, or 2.0 ng of PDGF arepresent after decellularization per gram of dried tissue filler (or anyvalue in between). In some embodiments, 0.05, 0.1, or 0.2 ng ofangiopoitin-2 are present after decellularization per gram of driedtissue filler (or any value in between) when the tissue filler is madefrom processed lung tissue. In some embodiments, 0.5, 1.0, or 1.5 ng offollistatin are present after decellularization per gram of dried tissuefiller (or any value in between).

In various embodiments, the decellularization process does not alter theextracellular matrix of the harvested tissue. For example, theextracellular matrix in decellularized tissue can remain substantiallyunaltered when compared to non-decellularized tissue. The extracellularmatrix can consist of collagen, elastin, fibronectin, and proteoglycans,among other extracellular proteins. In some embodiments, furtherproteolytic processing is employed to remove undesirable extracellularmatrix components. For example, alpha-galactosidase can be applied toremove alpha-galactose moieties, as described below.

In some embodiments, after decellularization, tissue is treated withDNase to remove cellular DNA. In further embodiments, approximately 10,20, 30, 40, or 50 units/ml of DNase are used (or any value in between).In certain embodiments, RNase is added to the DNase solution. In furtherembodiments, approximately 10, 20, 30, 40, or 50 units/ml of RNase areused (or any value in between). In some embodiments, at least oneantibiotic is added to the DNase and/or RNase solution that is appliedto decellularized tissue. Appropriate antibiotics may include, forexample, gentamicin, streptomycin, penicillin, and amphotericin. Infurther embodiments, the antibiotic is added at a concentration ofapproximately 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg/ml (or any valuein between). In various embodiments, treatment of decellularized tissuewith DNase, RNase and/or antibiotics can be for 1 hour, 2 hours, 5hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, or 5 days (or anytime period in between). Appropriate duration of application andeffective concentrations will depend on the type of tissue and on theDNase, RNase, and/or antibiotics selected to process the tissue.

In certain embodiments, the tissue filler is coated in at least onesubstance that can reduce inflammation and/or fibrosis afterimplantation into a recipient tissue. As used herein, “coating” a tissuefiller in at least one substance means contacting one or more surfacesof the tissue filler, or an internal portion of the tissue filler, withthe at least one substance, or a solution containing the substance. Infurther embodiments, the at least one substance is HA and/or decorin.For example, tissue fillers can be incubated in a solution containingapproximately 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7mg/ml, 8 mg/ml, 9 mg/ml, or 10 mg/ml of hyaluronic acid (or any value inbetween). Incubation can be for, e.g., 1, 2, 3, 4, 5, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36, or 48 hours (or anytime period in between.) After incubation, hyaluronic acid-coated tissuefillers are washed for, e.g., 1, 6, 12, 15, 20, 24, 36, or 48 hours (orany time period in between). After incubation and washing, tissuefillers are coated in hyaluronic acid at a concentration of, e.g., 0.5,1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg of hyaluronic acid pergram of tissue filler (or any value in between). In another example,tissue fillers can be incubated in a solution containing approximately0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7mg/ml, 0.8 mg/ml, 0.9 mg/ml, or 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml, 2.5mg/ml, or 3.0 mg/ml of decorin (or any value in between). Incubation canbe for, e.g., 1, 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 30, 36, or 48 hours (or any time period in between).After incubation, decorin-coated tissue fillers are washed for, e.g., 1,6, 12, 15, 20, 24, 36, or 48 hours (or any time period in between).After incubation and washing, tissue fillers are coated in decorin at aconcentration of, e.g., 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, or 0.6 mg ofdecorin per gram of tissue filler (or any value in between.)

In further embodiments, tissue fillers are treated withalpha-galactosidase to remove alpha-galactose (α-gal) moieties. In someembodiments, to enzymatically remove α-gal epitopes, after washing thetissue thoroughly with saline, the tissue may be subjected to one ormore enzymatic treatments to remove α-gal antigens, if present in thesample. In some embodiments, the tissue may be treated with anα-galactosidase enzyme to eliminate α-gal epitopes. In furtherembodiments, the tissue is treated with α-galactosidase at aconcentration of 0.2 U/ml prepared in 100 mM phosphate buffered salineat pH 6.0. In other embodiments, the concentration of α-galactosidase isreduced to 0.1 U/ml or increased to 0.3 or 0.4 U/ml (or any value inbetween.) In other embodiments, any suitable enzyme concentration andbuffer can be used as long as sufficient antigen removal is achieved. Inaddition, certain exemplary methods of processing tissues to reduce orremove alpha-1,3-galactose moieties are described in Xu et al., TissueEngineering, Vol. 15, 1-13 (2009), which is incorporated by reference inits entirety.

Alternatively, in certain embodiments animals that have been geneticallymodified to lack one or more antigenic epitopes may be selected as thetissue source for a tissue filler. For example, animals (e.g., pigs)that have been genetically engineered to lack the terminal α-galactosemoiety can be selected as the tissue source. For descriptions ofappropriate animals, see U.S. application Ser. No. 10/896,594 and U.S.Pat. No. 6,166,288, which are incorporated herein by reference in theirentirety.

In various embodiments, tissue fillers are processed to reduce bioburden(i.e., to reduce the number of microorganisms growing on the tissuefiller). In some embodiments, tissue fillers are processed to removesubstantially all bioburden (i.e., to sterilize the tissue filler).Suitable bioburden reduction methods are known to one of skill in theart, and may include irradiation. Irradiation may reduce orsubstantially eliminate bioburden. In further embodiments, 15-17 kGyE-beam radiation is used. In other embodiments, tissue fillers arecoated in chlorhexidine (CHX) or silver to reduce or prevent bioburden.As used herein, “coating” a tissue filler in CHX or silver meanscontacting one or more surfaces of the tissue filler, or an internalportion of the filler, with CHX or silver or a solution containing CHXor silver. In certain embodiments, the concentration of CHX or silver isadjusted to remove substantially all bioburden. As used herein,“substantially all bioburden” means tissue fillers in which theconcentration of microorganisms growing on the filler is less than 1%,0.1%, 0.01%, 0.001%, or 0.0001% of that growing on untreated fillers.Effective concentrations of CHX may include 0.1 mg, 0.5 mg, 0.7 mg, 0.9mg, 1 mg, 1.5 mg, 2.0 mg, 2.5 mg, or 3.0 mg per gram of tissue filler(or any value in between). In some embodiments, the tissue filler may becoated with a solution containing silver at any concentration between 10μg/ml to 500 μg/ml of solution. Effective concentrations of silver mayinclude 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg,0.9 mg, or 1.0 mg per gram of tissue filler (or any value in between).

In various embodiments, processed tissue fillers are cut or molded intodesired shapes. Shapes may be selected to conform to the contours of thetissue into which the filler will be implanted, or to conform to thecontours of the void left by removal of native tissue. For example,tissue fillers may be cut into strips or molded into balls. Optimalshapes will be familiar to one of skill in the art and will depend onthe specific tissue into which a filler is being implanted and on thesize and/or shape of the bulk tissue that has been removed. For example,the desired shape of the tissue filler may depend on the shape and sizeof a tumor that has been removed from a patient.

In various embodiments, tissue fillers are cryopreserved for storage byfreeze-drying. In one example, the tissue filler is placed in acryopreservation solution that includes an organic solvent or water, toprotect against damage during freeze drying. In further embodiments,following incubation in the cryopreservation solution, the tissue filleris placed in a sterile vessel that is permeable to water vapor butimpermeable to bacteria. The vessel is cooled to a low temperature at aspecified rate that is compatible with the specific cryoprotectantformulation to minimize the freezing damage. The tissue filler is thendried at a low temperature under vacuum conditions. At the completion ofthe drying, the vacuum of the freeze drying apparatus is reversed with adry inert gas such as nitrogen, helium or argon. The tissue filler isthen sealed in an impervious container and stored until use. While theexample above describes one method for cryopreservation, one of skill inthe art will recognize that other such methods are well known in the artand may be used to cryopreserve and store tissue fillers.

In some embodiments, a tissue filler that has been freeze dried isrehydrated prior to implantation in a tissue. In further embodiments,the rehydrated tissue filler retains the spongy character and alsosubstantially the same stretchability and compressibility as found intissue filler that has not been freeze-dried. In certain embodiments,rehydrated tissue filler derived from decellularized porcine lungretains substantially the same shape and spongy character as found intissue filler that has not been freeze-dried (see FIG. 4).

The following examples serve to illustrate, and in no way limit, thepresent disclosure.

EXAMPLES Example 1 Decellularization of Porcine Lung and Liver

To obtain lung or liver scaffolds, tissue was harvested from 3-6month-old pigs in the slaughterhouse and shipped immediately to thelaboratory for processing. Porcine tissue was freeze-thawed twice at−80° C. and then washed with distilled water for 2 days. The bronchi orlarge blood vessels were removed by manual dissection. Porcine lung andliver tissues were decellularized at room temperature (22 to 25° C.) for5 days in a 10 mM HEPES buffer solution (pH 8.0) containing 2% sodiumdeoxycholate, 0.1% (w/v) Triton x-100 and 10 mM EDTA. Bottles weregently agitated on a shaker. Decellularized organ matrices were washedwith 0.9% saline to remove detergent until foam was no longer observedin solution. Tissues were then treated at room temperature (22 to 25°C.) for 24 hours in a second HEPES buffer solution (10 mM, pH 7.4)containing 30 units/ml DNase, 50 μg/ml gentamicin, 20 mM calciumchloride and 20 mM magnesium chloride. The DNase solution was discarded,and tissue was washed three times with 0.9% saline (30 min per wash).Decellularized tissues were further treated in phosphate-buffered saline(pH 6.5) containing 0.2 unit/ml α-galactosidase and 50 mM EDTA. Tissueswere sterilized with 0.1% PAA and 15-17 kGy E-beam. Tissues were storedin hydrated form or freeze-dried.

Example 2 Evaluation of Acellular Porcine Organ Matrices

To confirm the removal of cellular components, decellularized tissueswere digested with proteinase K and DNA content was measured by Quant-iTPicoGreen dsDNA Kit (Molecular Probes, Inc.), following themanufacturer's instructions. Decellularized tissues were also processedfor histology (H&E, Verhoeff's, Alcian blue) and evaluated using ascanning electron micrograph (SEM). Histological evaluation (H&E stain)demonstrated that the decellularization process completelydecellularized porcine organs while preserving a well-organized collagennetwork. FIGS. 1A & 1B show that the well-organized collagen network inporcine lung was preserved by the decellularization process. FIG. 1Cshows that processed porcine lung contains abundant elastin (Verhoeff'sstaining) and FIG. 1D shows the well-organized sponge ultrastructure ofthe decellularized lung (SEM).

To further characterize the organ matrices, extracellular matrixmolecules (ECM) were measured using anti-collagen type I, III, IV,fibronectin and laminin antibodies. FIGS. 2A, 2B & 2C show thatdecellularized porcine lung tissue contains some type I collagen andabundant type IV collagen and fibronectin. FIG. 2D shows that type IIIcollagen, as well as laminin (data not shown), is not a major componentof the acellular lung matrix. Evaluation of fresh porcine lung tissuewith the above antibodies demonstrated similar staining patterns,suggesting that the tissue processing method preserved the structure ofnative lung tissue matrices.

Example 3 Bioburden Quantification

TABLE 1 Samples CFU (Processed) CFU (Sterilized) Porcine Liver 2.85 ×10⁴ 0 Porcine Lung >10⁶ 0

To ensure matrix sterility, the bioburden of tissue matrices wasmeasured before and after a sterilization step. Table 1 shows theBioburden quantification of acellular porcine liver and lung matricesbefore and after sterilization. The bioburden was extracted from tissuematrices in 50 ml saline solution followed by stomacher 400 circulatorat 150 rpm for 2 minutes and collection on a 0.45 micron filter. Thefilter was placed on solid growth media and incubated at 37° C. for 3days to allow for microbial colony formation. The microbial colonieswere then counted to quantify the microorganisms in terms of colonyforming units (CFU). Table 1 shows that no bioburden was detected aftersterilization.

Acellular lung and liver matrices were also coated with theantimicrobial reagent Chlorhexidine (CHX) or silver. CHX is anantimicrobial used in many consumer products, such as mouthwash andcontact lens solutions. The concentration of CHX coated on the acellularlung and liver matrices was determined by high performance liquidchromatography (HPLC) to be 1.0 mg per gram of tissue. The concentrationof silver coated on the acellular matrices was determined by ICP to be0.22 mg per gram of tissue. Tissue matrices coated with CHX weremeasured for bioburden. Table 2 shows that the CHX coating efficientlyreduced bioburden.

TABLE 2 Samples CFU (Processed) CFU (Sterilized) Porcine Liver 2.85 ×10⁴ 0 Porcine Lung >10⁶ 0

Example 4 Mechanical Property of Porcine Lung and Liver

The softness of acellular porcine lung or liver tissues was measuredusing a durometer (Table 3). A durometer measures the indentationresistance of elastomeric or soft materials based on the depth ofpenetration of a conical indenter. Hardness values range from 0 to 100.A lower number indicates a softer material, whereas a higher numberindicates that the material is harder. The data from the durometer showsthat porcine lung and liver have similar softness to human breasttissue.

TABLE 3 Porcine Porcine Porcine Porcine Human breast muscle liver lungdermis (neck) tissue 29.4 ± 4.9 2.33 ± 0.92 5.63 ± 0.91 40.0 ± 8.6 5* (N= 4) (n = 7) (n = 7) (N = 24) *Reference

Example 5 Tissue Integrity Testing

To measure the retention of matrix integrity in processed lung and livertissue, glycosaminoglycan concentrations were analyzed by a SulfatedGlycosaminoglycan Assay (Biocolor Ltd.) following the manufacturer'sinstructions (FIG. 5). Porcine acellular liver and lung matrices containabundant GAG when compared to porcine acellular dermal tissue (PADM).

The effect of tissue processing on collagen stability was measured bymodulated temperature differential scanning calorimetry (TA Instruments,New Castle, Del.), as previously described. Gouk et al., J. Biomed. Mat.Res. Part B: Appl. Biomat. 84B: 205-217 (2007). The onset temperature(T_(m)) and enthalpy (ΔH) of collagen denaturation were determined byanalysis of thermograms using Universal Analysis 2000 software (version4.0) with dry tissue samples (FIG. 6). Thermograms of both porcine lungand liver acellular tissue matrices had unfolding onset temperatures forcollagen molecules around 59-60° C., and peak unfolding temperaturearound 62-64° C. The data demonstrate that the integrity of native lungand liver matrices is preserved after processing.

The susceptibility of collagen in lung and liver matrices to enzymedigestion was evaluated in vitro by type I collagenase (Sigma-Aldrich,St. Louis, Mo.) and proteinase K (Fisher Scientific, Fair Lawn, N.J.)following the protocol described previously (Gouk, 2007). Briefly,samples were incubated with collagenase for varying lengths of time at37° C. The digested tissues were then washed and freeze-dried. Theresistance to enzyme digestion was calculated as a percentage of the dryweight of tissue remaining at various time points (FIG. 7). FIG. 7suggests that liver and lung acellular matrices are more resistant tocollagenase digestion when compared to porcine dermal tissue.

Example 6 Anti-Fibrosis Coatings on Acellular Lung and Liver Matrices

To reduce the possibility of fibrosis tissue formation, acellular tissuematrices were coated with anti-fibrosis reagents, such as hyaluronan orrecombinant human decorin. For hyaluronan coating, tissue matrices wereincubated with 5 mg/ml of hyaluronic acid (HA) sodium salt (Fluka 53747)at room temperature for 16 hours. Binding of HA to tissue matrices wasconfirmed by Alcian blue staining (FIG. 8). FIG. 8 shows that lungmatrices were successfully coated with HA. The concentration of HAcoated on the tissue matrices was determined by a DMMB colorimetricassay. Briefly, the HA coated tissue matrices were digested withcollagenase and the supernatants were quantified to determine the GAGconcentration using dimethylmethylene blue (DMMB) staining. FIG. 9 showsthat HA was effectively coated onto the tissues and persisted afterwashing overnight with 3 changes.

For decorin coating, tissue matrices were incubated with 1 mg/ml ofrecombinant human decorin (DCN) at room temperature for 16 hours andthen washed overnight to remove unbound decorin. Binding of humandecorin to the tissue matrices was confirmed by an anti-human decorinantibody that does not react with porcine decorin (FIG. 10). The decorinconcentration coated on the tissue was determined with a RayBio HumanDecorin ELISA kit (Ray Biotech Inc). FIG. 11 shows that human decorinwas effectively coated onto liver matrices and persisted after washingovernight with 3 changes.

Example 7 In Vitro Cell Growth and Inflammation

Both rat fibroblast cells (ATCC, CRL-1213) and human fibroblast cells(ATCC, CRL-2522) were cultured in minimal essential medium (MEM)supplemented with 10% fetal bovine serum (ATCC, MD). Human monocyteswere cultured in macrophage-serum-free medium (SFM) (Gibco, CA). Ratbone marrow or adipose mesenchymal stem cells (MSC) were cultured in MSCexpansion medium (Millipore, MA). Acellular lung and liver tissues werewashed in saline (supplemented with 50 μg/ml gentamicin) for 12 hourswith shaking at room temperature (6 changes) and then placed at thebottoms of the wells in 24-well plates (0.5×0.5 cm of tissue per well).One milliliter of cells (5-10×10⁴ cells/ml) was applied to the tissues.Co-cultures of tissues with cells were then fixed and stained for H&E at1, 2 and 3 weeks of culture. FIG. 12 shows that porcine lung and livermatrices support growth, migration and proliferation of rat MSC andfibroblast cells.

Fresh human mononuclear cells were isolated from human donor blood byFicoll separation. One million cells in 1 ml macrophage-SFM (Gibco, CA)were applied to acellular tissue matrices (0.5×0.5 cm) placed at thebottoms of the wells in 24-well plates. The culture supernatant wascollected 7 days post co-culture and centrifuged. The supernatant wasanalyzed for cytokines (IL-1, -2, -4, -6, -8, -10, IFN-g and TNF-a) withan Elisa kit for human cytokines. FIG. 13 shows that porcine liver andlung matrices did not induce significant inflammatory cytokines when thematrices were cultured with human blood mononuclear cells.

Example 8 Growth Factors in Processed Lung or Liver Tissue Matrices

Growth factors retained in processed lung and liver tissue matrices weredetermined using a Bio-Plex Pro Assay (BioRad). Briefly, processedtissue was washed with saline and then freeze-dried and cryo-milled.Cryo-milled tissue (100 mg) was extracted in 1 ml tissue extractionreagent I (invitrogen) at 4° C. overnight. The supernatant was used forBio-Plex Pro Assays (Human Angiogenesis Panel). The data shows that thedecellularizing and processing of lung and liver tissues preserve thekey growth factors (FGF, VEGF, PDGF) found in porcine organ matrices(table 4).

TABLE 4 ng/g dried Angiopoitin- tissue FGF VEGF PDGF 2 FollistatinProcessed 18.43 2.04 0.72 undetectable 0.72 Liver Processed 42.7 8.441.89 0.11 0.58 Lung

Example 9 In Vivo Testing

To assess the biological response to processed tissue matrices in vivo,processed porcine lung and liver tissue matrices (1.0×1.0×0.5 cm) weresubcutaneously implanted in rats for 14 days. Processed acellularmatrices were compared to matrices coated with an anti-microbial agent,HA, or human decorin. The explants were grossly evaluated for theirshape, hardness, and size (Table 5 & FIG. 14). After two weeks,implanted porcine lung matrices had the same shape, size and spongeproperty as observed prior to implantation. CHX-coated liver tissuematrices were firm and seemed encapsulated, while HA or decorin-coatedliver tissue matrices were soft with less surrounding connectivetissues.

TABLE 5 Gross observation of the explants Shape Explants change HardnessSize Liver matrix No Slightly firm 1 × 1 × 0.6 cm Liver-CHX No Firm 1.5× 1 × 0.8 cm   Liver-HA No Soft 1 × 1 × 0.8 cm Liver-Decorin No Soft 1 ×1 × 0.8 cm

The processed porcine lung and liver tissue matrix explants were alsoevaluated histologically for cell infiltration, inflammation andencapsulation. Histology demonstrated cellular infiltration in allgrafts. Matrices coated with CHX appeared to be encapsulated and showedless fibroblast cell repopulation (FIG. 15). Matrices coated with HA anddecorin had minimal inflammation (FIG. 16). This data suggests thatcoating matrices in HA and human decorin reduces inflammation andencapsulation. Histology also demonstrated cellular repopulation andrevascularization in the implanted matrices. Human decorin-coated graftsappeared to have fewer fibroblast cells than did the uncoated orHA-coated grafts, indicating regulatory effects of decorin on fibroblastproliferation (FIG. 17).

To assess the host biological response to the implants, two weekexplants of acellular liver matrix grafts were immunostained withanti-vimentin to detect fibroblasts, anti-vWF to detect neo-vesselformation, and anti-alpha-smooth muscle cell actin to detectmyofibroblast cells (myofibroblast cells have been reported to beinvolve in fibrosis formation). Fibroblast cell repopulation wasconfirmed by immunostaining and neo-vessel formation was shown by vWFstaining (FIGS. 18A & B). SMC-α-Actin staining did not suggest inductionof myofibroblast cells (FIG. 18C). The grafts coated with human decorinappeared to have fewer fibroblast cells when compared to non-coated orHA-coated grafts (FIG. 19).

To further characterize the inflammatory cells in the liver and lunggrafts, T cells, B cells and Macrophages at the graft site were stainedusing antibodies. The level of inflammation was scored as follows:0=none, 1=mild, 2 moderate, 3=significant. The data in Table 6 indicatethat the porcine acellular lung and liver matrices do not inducesignificant inflammation in a rat subcutaneous implantation model.

TABLE 6 T cells B cells Macrophages Lung 1 1 1 Lung-HA 0.5 0.5 0.5Lung-decorin 0.5 0.5 0.5 Liver 1 1 1 Liver-HA 0.5 0.5 0.5 Liver-decorin0.5 0.5 0.5

To assess if coated decorin remained on acellular matrices over time,acellular liver matrices coated with decorin were stained withanti-human decorin antibody two weeks after rat subcutaneousimplantation (FIG. 20). The results suggest that coated human decorinwas present on the implant 2-weeks post-implantation.

The preceding examples are intended to illustrate and in no way limitthe present disclosure. Other embodiments of the disclosed devices andmethods will be apparent to those skilled in the art from considerationof the specification and practice of the devices and methods disclosedherein.

What is claimed is:
 1. A tissue filler, comprising: an acellular tissuematrix having a stable three-dimensional structure, having the abilityto maintain the same shape, size, and sponge property after beingimplanted for two weeks as was observed prior to implantation; and atleast one of exogenous hyaluronic acid (HA) and exogenous decorin on asurface of the acellular tissue matrix at a concentration which reducesan inflammatory response or fibrosis when the tissue filler is implantedin a body as compared to the same tissue filler not comprisinghyaluronic acid or decorin.
 2. The tissue filler of claim 1, wherein aconcentration of HA on the acellular tissue matrix is betweenapproximately 0.5 mg and approximately 5.0 mg per gram of tissue filler.3. The tissue filler of claim 1, further comprising at least one growthfactor.
 4. The tissue filler of claim 3, wherein the at least one growthfactor is selected from at least one of FGF, VEGF, PDGF, Angiopoitin-2,or Follistatin.
 5. The tissue filler of claim 1, wherein the tissuefiller has been treated to reduce a bioburden.
 6. The tissue filler ofclaim 1, further comprising an antimicrobial agent.
 7. The tissue fillerof claim 6, wherein the antimicrobial agent includes at least one ofchlorhexidine (CHX) and silver.
 8. The tissue filler of claim 7, whereinthe CHX has a concentration between approximately 0.1 mg andapproximately 3.0 mg per gram of tissue filler.
 9. The tissue filler ofclaim 8, wherein the silver has a concentration between approximately0.1 mg and approximately 1.0 mg per gram of tissue filler.
 10. Thetissue filler of claim 1, wherein the tissue filler is capable of beingcompressed up to approximately ⅔ of its length or width.
 11. The tissuefiller of claim 10, wherein the tissue filler is capable of returning toits original dimensions after release of compression.
 12. The tissuefiller of claim 1, wherein a concentration of decorin on the acellulartissue matrix is between approximately 0.3 mg and approximately 1.0 mgper gram of tissue filler.
 13. The tissue filler of claim 1, wherein theacellular tissue matrix comprises a dermal matrix.
 14. A method oftreating a tissue site, comprising: selecting a tissue site; andimplanting the tissue filler of claim 1 in the tissue site.
 15. A tissuefiller, comprising: an acellular tissue matrix having a stablethree-dimensional structure, having the ability to maintain the sameshape, size, and sponge property after being implanted for two weeks aswas observed prior to implantation; and exogenous hyaluronic acid (HA)on a surface of the acellular tissue matrix at a concentration whichreduces an inflammatory response or fibrosis when the tissue filler isimplanted in a body as compared to the same tissue filler not comprisinghyaluronic acid.
 16. The tissue filler of claim 15, wherein aconcentration of HA on the acellular tissue matrix is betweenapproximately 0.5 mg and approximately 5.0 mg per gram of tissue filler.17. The tissue filler of claim 15, wherein the acellular tissue matrixcomprises a dermal matrix.
 18. A tissue filler, comprising: an acellulartissue matrix having a stable three-dimensional structure, having theability to maintain the same shape, size, and sponge property afterbeing implanted for two weeks as was observed prior to implantation; andexogenous decorin on a surface of the acellular tissue matrix at aconcentration which reduces an inflammatory response or fibrosis whenthe tissue filler is implanted in a body as compared to the same tissuefiller not comprising decorin.
 19. The tissue filler of claim 18,wherein a concentration of decorin on the acellular tissue matrix isbetween approximately 0.3 mg and approximately 1.0 mg per gram of tissuefiller.
 20. The tissue filler of claim 18, wherein the acellular tissuematrix comprises a dermal matrix.